High plasma lipid levels are a risk factor of atherosclerosis. Our goal is to understand lipoprotein assembly for which microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) are essential. We described a high affinity MTP binding site within amino acids 270-570 of apoB. Also, we identified a novel antagonist, AGI-S117, which inhibited apoB/MTP binding without affecting MTP's lipid transfer activity, and decreased apoB secretion in HepG2 and McA-RH7777 cells. We hypothesize that apoB/MTP binding is essential for lipoprotein assembly because it is crucial for apoB translation, translocation across endoplasmic reticulum membranes, and lipidation. We postulate that the disruption of this binding will increase the binding of heat shock protein 70 and ubiquitin, and proteosome- mediated contranslational degradation of apoB, and decreases MTP- mediated lipid transfer to apoB. Aim1: We will study the effect of AGI-S17 on the transcription, translation and post-translational degradation of apoB100 in HepG2 cells. These studies will test the hypothesis that AGI-S117 in hibis apoB translation and decreases its secretion. AIMII: We propose that a high affinity MTP binding site in apoB is required for lipoprotein assembly. To identify the binding site within amino acids 270-570 of apoB, we will perform deletion and site-directed mutagenesis, then transiently express mutant apoB polypeptides in COS-7 cells to assess their binding to MTP. Also, the synthesis and secretion of mutant apoB polypeptides that do not bind MTP will be studied in stably transfected McA-RH7777 cells. Aim M: We hypothesize that two crucial events, translocation and lipidation of apoB, require apoB/MTP interactions. The effect of inhibition of apoB/MTP binding on the translocation, association with heat show protein 70 and ubiquitin, and cotranslational degradation of apoB will be studied in HepG2 cells treated with AGI-S17, and in McA-RH7777 cells expressing mutant apoB polypeptides. Thee role of apoB/MTP binding in the transfer of lipids to apoB will be studied in vitro using AGI-S17. These studies should show that apoB/MTP binding is important in early steps of lipoprotein assembly. Our future goal is to assess therapeutic efficacy of AGI-S17 in decreasing lipid levels in hyperlipidemic patients.